Hi,

The quality of the feeder layer does not look good.

You can try to increase the number/viability of the iPSC colony by using: - serum-free supplement CloneR; - RevitaCell ™ supplement or - ROCK inhibitor.

Moreover, the TeSR and E8/E8 flex medium should be used with vitronectin.

Good luck!

Justyna Augustyniak

Hi Justyna Augustyniak

Thanks for your reply.

Why do you say that feeder layer does not look good? These are commercial irrMEF from gibco.

In the cytotune protocol they state that vitronectin can be replaced by Geltrex in combination with E8 medium...

Thanks

Sofia

Hi,

It is easier to passage iPSC cells in E8 medium (in colonies) with 0.5 mM EDTA on vitronectin. EDTA is the most gentle method of iPSC passaging.

Geltrex and Matrigel better work with ESC medium were iPS cells are passage mostly with Trypsin, Triple or dispase were cells as a single cell.

What kind of medium do you use when you seed the MEFs on gelatin? What is the composition of this medium proposed by the company that sells MEFs?

In which density you seed MEFs on gelatin?

Justyna

Hi Justyna Augustyniak I lost your comment.

I use the exact MEF medium they recommend in the Cytotune protocol:

DMEM

10% FBS

1% NEAA

100 uL bME.

And I seed 250.000 cells/well (6 well plate)

Sofia

Hi Sofia Melo Calado Sofia, how do you induce your PBMC's to obtain pluripotency?

Hi Anna Knyazer .

I use the cytotune reprogramming kit.